J. Agrie. Food Chem. 1991, 39, 1029-1032
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Electrophoretic, Solubility, and Functional Properties of Commercial Soy Protein Isolates Estela L. Arrese,* Delia A. Sorgentini,1 Jorge R. Wagner,1 and Maria C. Añón’-1 1
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Centro de Investigación y Desarrollo en Criotecnologia de Alimentos (CIDCA), Facultad de Ciencias Exactas, UNLP, Calle 47 y 116, 1900 La Plata, Argentina, and Cátedra de Química Biológica, Facultad de Ciencias Exactas, UNLP, Calle 47 y 115, 1900 La Plata, Argentina
The effect of protein composition and degree of protein denaturation on the solubility, water-imbibing capacity (WIC), viscosity, and gelation capacity of commercial soy protein isolates was studied. It was found that the degree of denaturation may affect protein solubility, but very denatured proteins with high solubility were also detected. Isolates containing completely denatured proteins showed low gelation capacity. This characteristic is closely related to the relative amounts of the 7S and 11S proteins, since ß-lS subunit and basic 11S polypeptide were present in decreased concentrations in the soluble protein fraction. Isolates with a high degree of denaturation and intermediate solubility values presented the maximal WIC. Results confirmed that the apparent viscosity of soy protein dispersions is intimately related to WIC.
soluble protein. Protein solubility was expressed as the ratio of soluble to total protein. Total protein of samples was determined by the Kjeldahl method (N X 6.25) and the soluble protein by the biuret method (Gornall et al., 1949). Degree of Denaturation. Differential scanning calorimetry (DSC) was used to assess the degree of denaturation of commercial protein isolates. DSC thermograms were recorded on a Du Pont Model 910 calorimeter. Heating rate was 10 °C min"1. Samples (15-20 mg) of 20% (w/v) dispersions in distilled water were hermetically sealed in aluminum pans; a double empty pan was used as reference. After DSC analysis, the pans were punctured and the dry matter content was determined by drying overnight at 105 °C. Peaks indicating an endothermic heat flow were obtained for scans of some samples. To determine peak areas, baselines were constructed as shown in Figure 1 and the area obtained by using a Morphomat 34 Zeiss image analyzer. The enthalpy of total denaturation ( ) was calculated according to the method of Arntfield and Murray (1981), and the values were expressed in calories per gram of dry weight basis. Triplicate samples were evaluated by DSC. Electrophoresis (SDS-PAGE). Slab sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the discontinuous buffer system of Laemmli (1970) at a gel concentration of 12.6% using Pharmacia gel electrophoresis apparatus GE-2/4. Gel slabs were fixed and stained simultaneously in a solution of methanol, acetic acid, and water (5:5:2) and 0.1% Coomassie Brilliant Blue R-250. Molecular weights of the protein bands were estimated by means of a MWSDS-70L kit (Sigma Chemical Co.). Analyses were done in duplicate. Sample preparation for SDS-PAGE was carried out as follows: (a) 30 mg of protein isolate was dissolved in 30 mL of 0.086 M Tris, 0.09 M glycine, and 4 mM EDTA, pH 8 buffer, containing 8 M urea (urea buffer); (b) 30 mg of protein isolate was shaken in 30 mL of distilled water at 20 °C for 1 h and centrifuged (12100g, 4 °C, 20 min). The dispersions (a) and the supernatants (b) were mixed with an equal volume of SDS-PAGE sample buffer containing 5% (v/v) 2-mercaptoethanol. About 50 ug of protein was applied to each gel slot. For protein quantification by densitometric scanning, gels were scanned at 570 nm (reference 405 nm) by using Shimadzu dual-wavelength TLC scanner CS-910. The areas corresponding to each band were measured by using image analyzer Morphomat 34 Zeiss. For this purpose, each peak was determined by vertical lines perpendicular to the baseline of the densitogram. Total 7S and US percentages were calculated as the sum of the areas of their subuni ts/polypeptides with respect to the total area of the densitogram. The quantitative estimation of each subunit/ polypeptide of
INTRODUCTION One of the most popular plant protein sources to serve ingredient in food formulation is soy protein. The commercial preparation of soy proteins causes physical and chemical changes that affect their functional properties (Kinsella, 1979). These changes occur to a varying extent; thus, each preparation has to be evaluated with regard to functional properties. Globulins glycinin (11S) and d-conglycinin (7S) are the major components of soy isolates. The former has an estimated molecular weight (MW) of 309 000-393 000 and consists of acidic polypeptide chains (A) (MW 37 000-40 000) and basic polypeptide chains (B) (MW 19 900-20 000) (Nielsen, 1985), while 7S has a trimeric structure having a molecular weight of 140 000-170 000 and consists of subunits a! (83 00057 000), a (76 000-57 000), and ß (53 000-42 000) (Brooks and Morr, 1985). These two globulins have different structures and molecular properties (Derbyshire et al., 1976; Kinsella et al., 1985) and different functional properties (Saio and Watanabe, 1978). The aim of this study was to determine (1) the degree of protein denaturation and the relative amounts of the different protein species and (2) the incidence of the structural features of as an
the proteins on the solubility, water-imbibing capacity, viscosity, and gelation capacity of commercial soy protein isolates.
MATERIALS AND METHODS
Materials. Nineteen commercial soy isolates were utilized; 1-15 were produced by Sanbra S. A. and 16-19 by Ralston Purina. They are isolates 1-4, Proteinmax 90 HG, differents lots; isolates 13 and 14, Proteinmax MP, differents lots; isolates 15 and 16, Supro 500 E, differents lots; isolate 17, Supro 610; isolate 18, Supro 515; and isolate 19, Supro 590. These isolates exhibited the following characteristics: range of pH of 1 % isolate dispersions in distilled water was 6.8—7.0; the total protein content expressed as wet basis was 83-90% and moisture 5.0-6.5%. Methods. Solubility. Isolate dispersions (1% w/v) were prepared in distilled water, stirred magnetically at 20 °C for 1 h, and then centrifuged at 12100g for 20 min. Aliquots from the supernatant were taken for determination of the contents of 1 1
CIDCA. Cátedra de Química Biológica. 0021-8561/91/1439-1029S02.50/0
©
1991 American Chemical Society
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Arrese et al.
J. Agríe. Food Chem.. Vol. 39, No. 6, 1991
Figure 1. Differential scanning calorimetry (DSC) thermograms of 20% (w/v) commercial soy protein isolates in distilled water with different degrees of denaturation. Sensitivity was 0.021 meal s'1 and heating rate 10 °C min'1. Baseline is shown in sample 1. DW, dry weight. 7S and 11S proteins was calculated as the percentage of the area of the subunit polypeptides with respect to the total 7S or US area.
Apparent Viscosity. Apparent viscosities (ijapp) were measured in isolate dispersions between 2.5 and 14% (w/w). Measurements were carried out at 20 °C in a Haake Rota visco RV2 viscometer using a Sensor system NV and a rotor speed varying from 0 to 128 rpm in 2 min. Apparent viscosity at 128 rpm was calculated as
Figure 2. Densitometric scans of the electrophoretic patterns: (a) Sample
=
Vapp
GS /
n
where G is an instrument factor (centipoise/scale grade min), S is the scale value, and n is the rotor speed (rpm). Imbibed Water Ratio. Ratios of total to imbibed (T/ ) water were calculated (Urbanski et al., 1983) as
T /
of total water/g of dispersion g of imbibed water/g of dispersion
P P(WIC) where P is grams of isolate per gram of dispersion and WIC is grams of water imbibed per gram of soy protein isolate. Gel Viscosity. Gel properties of the isolates were measured in 10% (w/w) dispersions in distilled water, heated at 80 °C for 30 min and then cooled overnight at 4 °C. Gel viscosity was measured at 25 °C in a Brookfield viscometer RVT using the Helipath stand with the T spindle series at 5 rpm. Values were expressed in poises. Water-Imbibing Capacity [WIC). The WIC of soy protein isolates was determined by using a modification of the Baumann apparatus (Torgensen and Toledo, 1977). This apparatus consists of a funnel connected to a horizontal capillary. A 50-mg sample was dusted on a wetted filter paper which was fastened to a glass filter placed on top of the funnel filled with water. The apparatus was kept at 20 °C. The uptake of water by the sample at equilibrium was read in the graduated capillary and expressed as milliliters of water imbibed per gram of isolate. Determinations were performed in duplicate. _
g
1
solubilized in
urea
4, 11, 14, and 17 water-soluble
(centipoise)
_
1
-
RESULTS AND DISCUSSION
Solubility and Denaturation Degree. Three thermograms corresponding to isolates having marked differences in their degrees of denaturation are shown in Figure 1. Peaks corresponding to endothermic transitions are attributed to 7S (Tmax 74 °C) and 11S (Tmax 83 °C) proteins (Hermansson, 1978). The AH value represents a valuable parameter in assessing the degree of denaturation of plant proteins (Hermansson, 1978; Murray et al., = 1981). Thus, isolate 17 is completely denatured ( 0 cal/g) and isolate 5 is partially denatured. Values of total enthalpy of denaturation ( ) of all samples are shown in Table I. Isolates having maximal values present solubility values greater than 50%, while those
buffer; (b, c, d, e) soy isolates fractions, respectively.
Table I. Solubility and Total Denaturation Enthalpy ( ) of Commercial Soy Isolates soy
solubility,
AHj,
isolates
%
cal/g
83.6 73.8 54.7 57.8 68.1 61.0 44.0 36.3 58.0 48.1
2.85 2.53 2.30 2.16 0.60 0.65 0.45 0.30 0.085 0.081
1
2 3
4 5
6 7
8 9 10
soy isolates
solubility,
,
%
cal/g
11 12 13
59.0 83.8 45.5 27.7 29.7 27.9 24.8 20.6 36.5
0.30 0.10
14 15 16 17 18 19
0 0 0 0 0 0 0
= 0 isolates with cal/g have solubilities below 50%. Bearing in mind that denaturation of globular proteins leads to unfolding of the polypeptide chain, hydrophobic groups located in the interior of the molecule would be exposed for interaction with water, thus leading to a decrease in solubility. Therefore, solubility could serve as an index of denaturation for these proteins. At variance with this, some isolates with a high degree of denaturation (5, 6, 9,11, and 12), also have high solubilities (Table I). These cases, already reported by Shen (1976) and Hermansson (1986), indicate that it is not feasible to correlate low solubility with protein denaturation in commercial soy protein isolates. Therefore, determinations of solubility as a single index parameter are not a sensitive indicator of denaturation. Other aspects should be analyzed to understand the cause of this behavior. For instance, it would be interesting to study the superficial hydrophobicity of different isolates as well as the presence of water-soluble aggregates. Relative Composition of Different Protein S pecies. The proportion of 7S and 11S proteins present in commercial isolates was evaluated by means of SDS-PAGE of samples solubilized in urea buffer. The densitogram corresponding to sample 1 is shown in Figure 2a. All other isolates showed similar electrophoretic patterns. Molec-
J. Agrie. Food Cham., Vol. 39, No. 6, 1991
Properties of Soy Protein Isolates
Table II.
Quantitative Estimation of
7S
Protein Subunits and 1 IS Protein Polypeptides of Water-Soluble
isolates
I, AH >
II,
•
1-4
2
